Microscale Neuronal Activity Collectively Drives Chaotic and Inflexible Dynamics at the Macroscale in Seizures.

Neuronal activity propagates through the network during seizures, engaging brain dynamics at multiple scales. Such propagating events can be described through the avalanches framework, which can relate spatiotemporal activity at the microscale with global network properties. Interestingly, propagating avalanches in healthy networks are indicative of critical dynamics, where the network is organized to a phase transition, which optimizes certain computational properties. Some have hypothesized that the pathologic brain dynamics of epileptic seizures are an emergent property of microscale neuronal networks collectively driving the brain away from criticality. Demonstrating this would provide a unifying mechanism linking microscale spatiotemporal activity with emergent brain dysfunction during seizures. Here, we investigated the effect of drug-induced seizures on critical avalanche dynamics, using in vivo whole-brain two-photon imaging of GCaMP6s larval zebrafish (males and females) at single neuron resolution. We demonstrate that single neuron activity across the whole brain exhibits a loss of critical statistics during seizures, suggesting that microscale activity collectively drives macroscale dynamics away from criticality. We also construct spiking network models at the scale of the larval zebrafish brain, to demonstrate that only densely connected networks can drive brain-wide seizure dynamics away from criticality. Importantly, such dense networks also disrupt the optimal computational capacities of critical networks, leading to chaotic dynamics, impaired network response properties and sticky states, thus helping to explain functional impairments during seizures. This study bridges the gap between microscale neuronal activity and emergent macroscale dynamics and cognitive dysfunction during seizures.SIGNIFICANCE STATEMENT Epileptic seizures are debilitating and impair normal brain function. It is unclear how the coordinated behavior of neurons collectively impairs brain function during seizures. To investigate this we perform fluorescence microscopy in larval zebrafish, which allows for the recording of whole-brain activity at single-neuron resolution. Using techniques from physics, we show that neuronal activity during seizures drives the brain away from criticality, a regime that enables both high and low activity states, into an inflexible regime that drives high activity states. Importantly, this change is caused by more connections in the network, which we show disrupts the ability of the brain to respond appropriately to its environment. Therefore, we identify key neuronal network mechanisms driving seizures and concurrent cognitive dysfunction.

Bayesian inference of neuronal assemblies.

In many areas of the brain, both spontaneous and stimulus-evoked activity can manifest as synchronous activation of neuronal assemblies. The characterization of assembly structure and dynamics provides important insights into how brain computations are distributed across neural networks. The proliferation of experimental techniques for recording the activity of neuronal assemblies calls for a comprehensive statistical method to describe, analyze and characterize these high dimensional datasets. The performance of existing methods for defining assemblies is sensitive to noise and stochasticity in neuronal firing patterns and assembly heterogeneity. To address these problems, we introduce a generative hierarchical model of synchronous activity to describe the organization of neurons into assemblies. Unlike existing methods, our analysis provides a simultaneous estimation of assembly composition, dynamics and within-assembly statistical features, such as the levels of activity, noise and assembly synchrony. We have used our method to characterize population activity throughout the tectum of larval zebrafish, allowing us to make statistical inference on the spatiotemporal organization of tectal assemblies, their composition and the logic of their interactions. We have also applied our method to functional imaging and neuropixels recordings from the mouse, allowing us to relate the activity of identified assemblies to specific behaviours such as running or changes in pupil diameter.

Calcium imaging and dynamic causal modelling reveal brain-wide changes in effective connectivity and synaptic dynamics during epileptic seizures.

Pathophysiological explanations of epilepsy typically focus on either the micro/mesoscale (e.g. excitation-inhibition imbalance), or on the macroscale (e.g. network architecture). Linking abnormalities across spatial scales remains difficult, partly because of technical limitations in measuring neuronal signatures concurrently at the scales involved. Here we use light sheet imaging of the larval zebrafish brain during acute epileptic seizure induced with pentylenetetrazole. Spectral changes of spontaneous neuronal activity during the seizure are then modelled using neural mass models, allowing Bayesian inference on changes in effective network connectivity and their underlying synaptic dynamics. This dynamic causal modelling of seizures in the zebrafish brain reveals concurrent changes in synaptic coupling at macro- and mesoscale. Fluctuations of both synaptic connection strength and their temporal dynamics are required to explain observed seizure patterns. These findings highlight distinct changes in local (intrinsic) and long-range (extrinsic) synaptic transmission dynamics as a possible seizure pathomechanism and illustrate how our Bayesian model inversion approach can be used to link existing neural mass models of seizure activity and novel experimental methods.

Emergent properties of the optic tectum revealed by population analysis of direction and orientation selectivity.

How local circuits within the brain process visual information has classically been addressed at the single neuron level. Such reductionist approaches, however, struggle to capture the full scope of functional properties associated with even "simple" brain nuclei. Using population functional calcium imaging, we aim to describe how local circuits within the zebrafish optic tectum process visual information. Specifically, how are previously identified direction-selective (DS) and orientation-selective (OS) retinal ganglion cell (RGC) inputs (Nikolaou et al., 2012) represented in tectal cells? First, we identify an emergent population of DS tectal cell with a direction preference not explicitly present in any one of the RGC inputs. Second, this is associated with a striking shift from a tiled and triangular representation of directional space (RGC inputs) into an overlapping cardinal representation by tectal cell populations. Third, and in contrast, we find that orientation space is represented similarly in both the RGC input and tectal cell populations illustrating feature-dependent differences in how tectal circuits process their inputs. Finally, we identify OS and two populations of DS cells at the superficial border of the tectal neuropil, one of which is an emergent population. This study, together with our previous one (Nikolaou et al., 2012), demonstrate that direction-selectivity is established in both the retina and tectum.

A systems-based dissection of retinal inputs to the zebrafish tectum reveals different rules for different functional classes during development.

We have examined the form, diversity, and organization of three functional classes of retinal inputs to the zebrafish optic tectum during development. Our systems-based approach was to analyze data from populations of retinal ganglion cells labeled with a presynaptic targeted calcium indicator, synaptophysin GCaMP3 (SyGCaMP3). Collectively, our findings provide an insight as to the degree of visual encoding during retino-tectal development and how it dynamically evolves from a nascent and noisy presynaptic neural-scape to an increasingly complex and refined representation. We report five key features: (1) direction-selective inputs are developmentally invariant; (2) orientation-selective inputs exhibit highly dynamic properties over the same period, with changes in their functional characteristics and spatial organization; (3) inputs defined as anisotropic are an early dominant functional class, with heterogeneous response profiles, which progressively diminish in incidence and spatial extent; (4) dark rearing selectively affects the orientation-selective responses: both functional characteristics and relative spatial distributions; and (5) orientation-selective inputs exhibit four subtypes, two more than previously identified in any species. Our approach was to label RGC axon terminals with an indicator of activity and quantitatively characterize coherent response properties to different visual stimuli. Its application in the zebrafish, given its small size and the accessibility of the tectum, has enabled a quick yet robust assessment of multiple functional populations of responses.

Topographically Localized Modulation of Tectal Cell Spatial Tuning by Complex Natural Scenes.

The tuning properties of neurons in the visual system can be contextually modulated by the statistics of the area surrounding their receptive field (RF), particularly when the surround contains natural features. However, stimuli presented in specific egocentric locations may have greater behavioral relevance, raising the possibility that the extent of contextual modulation may vary with position in visual space. To explore this possibility, we utilized the small size and optical transparency of the larval zebrafish to describe the form and spatial arrangement of contextually modulated cells throughout an entire tectal hemisphere. We found that the spatial tuning of tectal neurons to a prey-like stimulus sharpens when the stimulus is presented against a background with the statistics of complex natural scenes, relative to a featureless background. These neurons are confined to a spatially restricted region of the tectum and have receptive fields centered within a region of visual space in which the presence of prey preferentially triggers hunting behavior. Our results suggest that contextual modulation of tectal neurons by complex backgrounds may facilitate prey-localization in cluttered visual environments.

Melatonin suppression does not automatically alter sleepiness, vigilance, sensory processing, or sleep.

Presleep exposure to short-wavelength light suppresses melatonin and decreases sleepiness with activating effects extending to sleep. This has mainly been attributed to melanopic effects, but mechanistic insights are missing. Thus, we investigated whether two light conditions only differing in the melanopic effects (123 vs. 59 lx melanopic EDI) differentially affect sleep besides melatonin. Additionally, we studied whether the light differentially modulates sensory processing during wakefulness and sleep. Twenty-nine healthy volunteers (18-30 years, 15 women) were exposed to two metameric light conditions (high- vs. low-melanopic, ≈60 photopic lx) for 1 h ending 50 min prior to habitual bed time. This was followed by an 8-h sleep opportunity with polysomnography. Objective sleep measurements were complemented by self-report. Salivary melatonin, subjective sleepiness, and behavioral vigilance were sampled at regular intervals. Sensory processing was evaluated during light exposure and sleep on the basis of neural responses related to violations of expectations in an oddball paradigm. We observed suppression of melatonin by ≈14% in the high- compared to the low-melanopic condition. However, conditions did not differentially affect sleep, sleep quality, sleepiness, or vigilance. A neural mismatch response was evident during all sleep stages, but not differentially modulated by light. Suppression of melatonin by light targeting the melanopic system does not automatically translate to acutely altered levels of vigilance or sleepiness or to changes in sleep, sleep quality, or basic sensory processing. Given contradicting earlier findings and the retinal anatomy, this may suggest that an interaction between melanopsin and cone-rod signals needs to be considered. Clinical Trial Registry: German Clinical Trials Register, DRKS00023602, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00023602.

Synaptic activity and activity-dependent competition regulates axon arbor maturation, growth arrest, and territory in the retinotectal projection.

In the retinotectal projection, synapses guide retinal ganglion cell (RGC) axon arbor growth by promoting branch formation and by selectively stabilizing branches. To ask whether presynaptic function is required for this dual role of synapses, we have suppressed presynaptic function in single RGCs using targeted expression of tetanus toxin light-chain fused to enhanced green fluorescent protein (TeNT-Lc:EGFP). Time-lapse imaging of singly silenced axons as they arborize in the tectum of zebrafish larvae shows that presynaptic function is not required for stabilizing branches or for generating an arbor of appropriate complexity. However, synaptic activity does regulate two distinct aspects of arbor development. First, single silenced axons fail to arrest formation of highly dynamic but short-lived filopodia that are a feature of immature axons. Second, single silenced axons fail to arrest growth of established branches and so occupy significantly larger territories in the tectum than active axons. However, if activity-suppressed axons had neighbors that were also silent, axonal arbors appeared normal in size. A similar reversal in phenotype was observed when single TeNT-Lc:EGFP axons are grown in the presence of the NMDA receptor antagonist MK801 [(+)-5-methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]. Although expansion of arbor territory is prevented when neighbors are silent, formation of transient filopodia is not. These results suggest that synaptic activity by itself regulates filopodia formation regardless of activity in neighboring cells but that the ability to arrest growth and focusing of axonal arbors in the target is an activity-dependent, competitive process.

Learning steers the ontogeny of an efficient hunting sequence in zebrafish larvae.

Goal-directed behaviors may be poorly coordinated in young animals but, with age and experience, behavior progressively adapts to efficiently exploit the animal's ecological niche. How experience impinges on the developing neural circuits of behavior is an open question. We have conducted a detailed study of the effects of experience on the ontogeny of hunting behavior in larval zebrafish. We report that larvae with prior experience of live prey consume considerably more prey than naive larvae. This is mainly due to increased capture success and a modest increase in hunt rate. We demonstrate that the initial turn to prey and the final capture manoeuvre of the hunting sequence were jointly modified by experience and that modification of these components predicted capture success. Our findings establish an ethologically relevant paradigm in zebrafish for studying how the brain is shaped by experience to drive the ontogeny of efficient behavior.

Targeted knockout of GABA-A receptor gamma 2 subunit provokes transient light-induced reflex seizures in zebrafish larvae.

Epilepsy is a common primary neurological disorder characterized by the chronic tendency of a patient to experience epileptic seizures, which are abnormal body movements or cognitive states that result from excessive, hypersynchronous brain activity. Epilepsy has been found to have numerous etiologies and, although about two-thirds of epilepsies were classically considered idiopathic, the majority of those are now believed to be of genetic origin. Mutations in genes involved in gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission have been associated with a broad range of epilepsy syndromes. Mutations in the GABA-A receptor gamma 2 subunit gene (GABRG2), for example, have been associated with absence epilepsy and febrile seizures in humans. Several rodent models of GABRG2 loss of function depict clinical features of the disease; however, alternative genetic models more amenable for the study of ictogenesis and for high-throughput screening purposes are still needed. In this context, we generated a gabrg2 knockout (KO) zebrafish model (which we called R23X) that displayed light/dark-induced reflex seizures. Through high-resolution in vivo calcium imaging of the brain, we showed that this phenotype is associated with widespread increases in neuronal activity that can be effectively alleviated by the anti-epileptic drug valproic acid. Moreover, these seizures only occur at the larval stages but disappear after 1 week of age. Interestingly, our whole-transcriptome analysis showed that gabrg2 KO does not alter the expression of genes in the larval brain. As a result, the gabrg2-/- zebrafish is a novel in vivo genetic model of early epilepsies that opens new doors to investigate ictogenesis and for further drug-screening assays.

In vivo imaging of synapse formation on a growing dendritic arbor.

The form of a neuron's dendritic arbor determines the set of axons with which it may form synaptic contacts, thus establishing connectivity within neural circuits. However, the dynamic relationship between dendrite growth and synaptogenesis is not well understood. To observe both processes simultaneously, we performed long-term imaging of non-spiny dendritic arbors expressing a fluorescent postsynaptic marker protein as they arborized within the optic tectum of live zebrafish larvae. Our results indicate that almost all synapses form initially on newly extended dendritic filopodia. A fraction of these nascent synapses are maintained, which in turn stabilizes the subset of filopodia on which they form. Stabilized filopodia mature into dendritic branches, and successive iterations of this process result in growth and branching of the arbor. These findings support a 'synaptotropic model' in which synapse formation can direct dendrite arborization.

Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms.

To explore the relationship between axon arbor growth and synaptogenesis, developing retinal ganglion cell (RGC) axon arbors in zebrafish optic tectum were imaged in vivo at high temporal and spatial resolution using two-photon microscopy. Individual RGC axons were dually labeled by expression of a cytosolic red fluorescent protein (DsRed Express) to mark arbor structure and a fusion of the synaptic vesicle protein synaptophysin with green fluorescent protein (Syp:GFP) to mark presynaptic vesicles. Analysis of time-lapse sequences acquired at 10 min intervals revealed unexpectedly rapid kinetics of both axon branch and vesicle cluster turnover. Nascent axonal branches exhibited short average lifetimes of 19 min, and only 17% of newly extended axonal processes persisted for periods exceeding 3 h. The majority (70%) of Syp:GFP puncta formed on newly extended axonal processes. Syp:GFP puncta also exhibited short average lifetimes of 30 min, and only 34% of puncta were stabilized for periods exceeding 3 h. Moreover, strongly correlated dynamics of Syp:GFP puncta and branch structure suggest that synaptogenesis exerts strong influences on both the extension and the selective stabilization of nascent branches. First, new branches form almost exclusively at newly formed Syp:GFP puncta. Second, stabilized nascent branches invariably bear Syp:GFP puncta, and the detailed dynamics of branch retraction suggest strongly that nascent synapses can act at branch tips to arrest retraction. These observations thus provide evidence that synaptogenesis guides axon arbor growth by first promoting initial branch extension and second by selective branch stabilization.

A Three-Layer Network Model of Direction Selective Circuits in the Optic Tectum.

The circuit mechanisms that give rise to direction selectivity in the retina have been studied extensively but how direction selectivity is established in retinorecipient areas of the brain is less well understood. Using functional imaging in larval zebrafish we examine how the direction of motion is encoded by populations of neurons at three layers of the optic tectum; retinal ganglion cell axons (RGCs), a layer of superficial inhibitory interneurons (SINs), and periventricular neurons (PVNs), which constitute the majority of neurons in the tectum. We show that the representation of motion direction is transformed at each layer. At the level of RGCs and SINs the direction of motion is encoded by three direction-selective (DS) subtypes tuned to upward, downward, and caudal-to-rostral motion. However, the tuning of SINs is significantly narrower and this leads to a conspicuous gap in the representation of motion in the rostral-to-caudal direction at the level of SINs. Consistent with previous findings we demonstrate that, at the level of PVNs the direction of motion is encoded by four DS cell types which include an additional DS PVN cell type tuned to rostral-to-caudal motion. Strikingly, the tuning profile of this emergent cell type overlaps with the gap in the representation of rostral-to-caudal motion at the level of SINs. Using our functional imaging data we constructed a simple computational model that demonstrates how the emergent population of PVNs is generated by the interactions of cells at each layer of the tectal network. The model predicts that PVNs tuned to rostral-to-caudal motion can be generated via convergence of DS RGCs tuned to upward and downward motion and feedforward tuned inhibition via SINs which suppresses responses to non-preferred directions. Thus, by reshaping directional tuning that is inherited from the retina inhibitory inputs from SINs can generate a novel subtype of DS PVN and in so doing enhance the encoding of directional stimuli.

Individual Neuronal Subtypes Exhibit Diversity in CNS Myelination Mediated by Synaptic Vesicle Release.

Regulation of myelination by oligodendrocytes in the CNS has important consequences for higher-order nervous system function (e.g., [1-4]), and there is growing consensus that neuronal activity regulates CNS myelination (e.g., [5-9]) through local axon-oligodendrocyte synaptic-vesicle-release-mediated signaling [10-12]. Recent analyses have indicated that myelination along axons of distinct neuronal subtypes can differ [13, 14], but it is not known whether regulation of myelination by activity is common to all neuronal subtypes or only some. This limits insight into how specific neurons regulate their own conduction. Here, we use a novel fluorescent fusion protein reporter to study myelination along the axons of distinct neuronal subtypes over time in zebrafish. We find that the axons of reticulospinal and commissural primary ascending (CoPA) neurons are among the first myelinated in the zebrafish CNS. To investigate how activity regulates myelination by different neuronal subtypes, we express tetanus toxin (TeNT) in individual reticulospinal or CoPA neurons to prevent synaptic vesicle release. We find that the axons of individual tetanus toxin expressing reticulospinal neurons have fewer myelin sheaths than controls and that their myelin sheaths are 50% shorter than controls. In stark contrast, myelination along tetanus-toxin-expressing CoPA neuron axons is entirely normal. These results indicate that while some neuronal subtypes modulate myelination by synaptic vesicle release to a striking degree in vivo, others do not. These data have implications for our understanding of how different neurons regulate myelination and thus their own function within specific neuronal circuits.

Lamination Speeds the Functional Development of Visual Circuits.

A common feature of the brain is the arrangement of synapses in layers. To examine the significance of this organizational feature, we studied the functional development of direction-selective (DS) circuits in the tectum of astray mutant zebrafish in which lamination of retinal ganglion cell (RGC) axons is lost. We show that although never laminar, the tuning of DS-RGC axons targeting the mutant tectum is normal. Analysis of mutant tectal neurons at late developmental stages reveals that directional tuning is indistinguishable from wild-type larvae. Furthermore, we show that structural plasticity of tectal dendrites and RGC axons compensates for the loss of lamination, establishing connectivity between DS-RGCs and their normal tectal targets. However, tectal direction selectivity is severely perturbed at earlier developmental stages. Thus, the formation of synaptic laminae is ultimately dispensable for the correct wiring of direction-selective tectal circuits, but it is crucial for the rapid assembly of these networks.

Neurobiology: imaging prey capture circuits in zebrafish.

Two recent studies used a virtual hunting assay and functional imaging to identify prey-capture circuits in zebrafish. Together they show that the optic tectum and a pretectal region are two retinorecipient areas important for the recognition and capture of prey.

Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

Fish vision: size selectivity in the zebrafish retinotectal pathway.

A recent study has shown that the zebrafish tectum processes inputs from the retina tuned to ethologically relevant size classes, suggesting a role for the tectum in selecting approach or avoidance behaviours based on size-based categorization of visual targets.

Functional imaging in the zebrafish retinotectal system using RGECO.

Genetically encoded calcium indicators (GECIs) allow repeated, non-invasive measurements of neural activity in defined populations of neurons, but until recently GECIs based on single fluorescent proteins have been limited to the green region of the color spectrum. Recent efforts in protein engineering have expanded the color palette of GECIs. One of these new GECIs, the red RGECO, is spectrally separate from the traditional GFP-based sensors such as GCaMP, and therefore opens the way for simultaneous, multicolor imaging of neural activity. While RGECO has been shown to report spontaneous calcium fluctuations in neurons, the precise relationship of RGECO signal to evoked-neural activity is not known. Measurements of neural activity using RGECO in vivo have also not been reported. Using dissociated hippocampal neurons we performed a systematic analysis of two forms of RGECO- a cytosolic form and a presynaptically localized form generated by fusion of RGECO to the presynaptic protein, synaptophysin (SyRGECO). We find that RGECO and GCaMP3 are comparable in terms of dynamic range, signal-to-noise ratios and kinetics but that RGECO is a more reliable reporter of single action potentials. In terms of performance SyGCaMP3 and SyRGECO are comparable, and both are more sensitive reporters of activity than the cytosolic form of each probe. Using the zebrafish retinotectal system we show that SyRGECO and RGECO are can report neural activity in vivo and that RGECO expression permits detailed structural analysis of neuronal arbors. We have exploited these attributes to provide a morphological and functional description of tectal cells selective for motion along the vertical axis. These results open up the possibility of using zebrafish to functionally image genetically defined pre- and postsynaptic circuit components, separable by color, which will be a powerful approach to studying neural interactions in the brain.

Teneurin-3 specifies morphological and functional connectivity of retinal ganglion cells in the vertebrate visual system.

A striking feature of the CNS is the precise wiring of its neuronal connections. During vertebrate visual system development, different subtypes of retinal ganglion cells (RGCs) form specific connections with their corresponding synaptic partners. However, the underlying molecular mechanisms remain to be fully elucidated. Here, we report that the cell-adhesive transmembrane protein Teneurin-3 (Tenm3) is required by zebrafish RGCs for acquisition of their correct morphological and functional connectivity in vivo. Teneurin-3 is expressed by RGCs and their presynaptic amacrine and postsynaptic tectal cell targets. Knockdown of Teneurin-3 leads to RGC dendrite stratification defects within the inner plexiform layer, as well as mistargeting of dendritic processes into outer portions of the retina. Moreover, a subset of RGC axons exhibits tectal laminar arborization errors. Finally, functional analysis of RGCs targeting the tectum reveals a selective deficit in the development of orientation selectivity after Teneurin-3 knockdown. These results suggest that Teneurin-3 plays an instructive role in the functional wiring of the vertebrate visual system.